Product Description
Reproducible and high-throughput in vitro models are essential for studying complex microbial communities. This kit is designed for the in vitro modeling of human airway mucus. Physiologic Air3Gel is presented here as a platform for culturing microbial samples. The protocol describes how to culture microbial samples in Physiologic Air3Gel using a syringe format and how to prepare the samples for downstream analyses. Downstream assays include cell viability assessment, flow cytometry, metabolomics, and sequencing. The potential applications of Physiologic Air3Gel include microbial mining, drug screening, and permeability assays.
Product Page: https://www.bac3gel.com/category/air3gel
Components
3x syringes, each containing 5 mL Physiologic Air3Gel.
50 mL Bac3Gel® dissolution medium (200 mM sodium citrate).
Material/Reagent Required But Not Provided
Syringe connector.
0.9% (w/v) NaCl solution prepared in deionized water.
Any required culture medium.
Culture medium is only required if the acquired Physiologic Air3Gel was not produced in any requested culture medium.
Storage Conditions
Store in a dry place inside tightly sealed containers at 2-8 °C.
This product can be stored for at least 9 months.
Precautions and Disclaimer
For R&D use only.
Procedure
Before you begin
The protocols below describe the specific steps for culturing single strain samples. However, these protocols have also been used to culture co-cultures, microbial consortia, and complex microbial samples. The Physiologic Air3Gel used in these protocols is composed of 20.0 mg.mL-1 porcine stomach type III mucin and 4.78 mg.mL-1 NaCl, respectively. Physiologic Air3Gel’s composition is fully tunable; customized compositions are commercially available (i.e. incorporation of other mucin types, proteins, lipids and media).
Ensure all instruments and consumables (pipettes, falcons, syringes, syringe connector, and microcentrifuge tubes) are sterilized and free of contaminants.
All manipulations should be performed under aseptic conditions, ideally in a Class II biological safety cabinet.
Prepare a 0.9% (w/v) sterile NaCl solution.
A. Preparation of Microbial Suspensions
1. Grow cultures of the species of interest (e.g. Pseudomonas aeruginosa) to the logarithmic (log) growth phase in the appropriate medium (e.g. LB medium).
Centrifuge the cultures to pellet the cells, then discard the supernatant.
Resuspend the cell pellet in LB medium or an appropriate culture medium.
If Physiologic Air3Gel was produced in culture medium, researchers should resuspend the cell pellet in 0.9 (w/v) NaCl instead of medium.
Measure the optical density at 600 nm (OD600) of the microbial suspension.
Adjust the cell concentration as needed.
B. Culturing in Physiologic Air3Gel
1. Discard the first 2-3 drops of Physiologic Air3Gel by gently pressing the syringe plunger.
2. Using the double syringe mixing method, combine the microbial suspensions prepared in A. and Physiologic Air3Gel in a 1:1 volume ratio by mixing 6-7 times.
- Dispense the syringe contents into a 15 mL falcon tube. Using a positive displacement pipette distribute the sample in equal volumes into microcentrifuge tubes.
If a syringe connector is not available, follow steps 3 and 4 instead, otherwise skip to step 5 after completing this step.
3. Dispense Physiologic Air3Gel from the syringe into a sterile 15 mL falcon tube.
4. Using a positive displacement pipette, transfer 500 μL of Physiologic Air3Gel into a microcentrifuge tube.
Instead of microcentrifuge tubes, researchers can use 24-well plates.
- Add an equal volume (500 μL) of the microbial suspensions prepared in A. and homogenize the mixture by gently pipetting.
5. Incubate the microcentrifuge tubes/24-well plate at 37 °C for up to 72 hours under aerobic or anaerobic conditions, according to the experimental design.
C. Dissolution of Physiologic Air3Gel
1. Add Bac3Gel® dissolution medium (200 mM sodium citrate solution) in a 1:1 ratio to each sample. Mix thoroughly to fully dissolve Physiologic Air3Gel structure.
- The homogenized samples can be used for downstream assays.
For sequencing: Centrifuge the samples (14,000 x g, 5 minutes), then use the pellet for DNA extraction.
For metabolomics: Centrifuge the samples (14,000 x g, 10 minutes), then collect and lyophilize the supernatant.
For measuring metabolic activity or optical density: Centrifuge the samples (14,000 xg, 5 minutes), discard the supernatant, then resuspend in 0.9 (w/v) NaCl.
Other applications include assessing cell viability, performing flow cytometry analysis, and conducting standard microbiological characterization assays.